Bone graft substitute composition

ABSTRACT

A bone graft substitute composition can include essentially of calcium sulfate, a mixing solution, and a plasticizing substance. A bone graft substitute composition can include calcium sulfate, demineralized bone matrix, cancellous bone, a plasticizing substance, and a mixing solution.

FIELD OF THE INVENTION

The invention relates to bone graft substitute compositions.

BACKGROUND

Calcium sulfate has been clinically used for many years as a bone voidfiller with successful results. A preliminary patentability searchproduced the following patents which appear to be relevant to thepresent invention:

Hanker et al., U.S. Pat. No. 4,619,655, issued Oct. 28, 1986, disclosesan animal implant comprising a scaffold material composed of plaster ofParis and a non-bioresorbable calcium material (such as calciumphosphate ceramic particles) bound with the plaster of Paris; a methodof inserting such a composition in fluid or semisolid form into theappropriate body location of an animal (e.g., about a fracture locus);and a method of inserting a preform of such composition into theappropriate location of an animal (e.g., at the locus of a fracture).

Gitelis, U.S. Pat. No. 5,147,403, issued Sep. 15, 1992, discloses amethod or technique for implanting a prosthesis comprising the steps offirst preparing the surface of a bone to receive the prosthesis, thenapplying a calcium sulfate suspension in free flowing form to theprepared bone surface, and then seating the prosthesis to the coatedbone surface.

Randolph, U.S. Pat. No. 5,614,206, issued Mar. 25, 1997, and U.S. Pat.No. 5,807,567, issued Sep. 15, 1998, disclose processes for preparingpellets by mixing of calcium sulfate, water and other medicaments toprovide controlled release of calcium sulfate and medicaments.

Snyder, U.S. Pat. No. 5,425,769, issued Jun. 20, 1995, discloses acomposition for an artificial bone substitute material consisting ofcollagen in a calcium sulfate matrix which can be rendered porous by afoaming agent. The composition is adaptable for osseous repair byadjusting the collagen and calcium sulfate in varying ratios to suitparticular applications and including admixtures of growth factors.

Sottosanti, U.S. Pat. No. 5,366,507, discloses a composition for use inbone tissue regeneration, the composition containing a barrier materialand a graft material. The barrier material can be calcium sulfate, whilethe graft material may consist of a composite graft material containingdemineralized, freeze-dried, allogeneic bone and calcium sulfate.

Sottosanti, U.S. Pat. No. 5,569,308, discloses a method for use in bonetissue regeneration including first filling a graft site with graftmaterial, and then placing a layer of barrier material over at least aportion of the graft material. The barrier material can be calciumsulfate, while the graft material may consist a composite graft materialcontaining demineralized, freeze-dried, allogeneic bone and calciumsulfate.

Hanker et al, “Setting of Composite Hydroxylapatite/Plaster Implantswith Blood 5 for Bone Reconstruction,” Proceedings of the 44th AnnualMeeting of the Electron Microscopy Society of America, Copyright 1986,discloses using blood as the only moistening agent in a plaster orplaster/HA mixture as long as accelerator salts are utilized, andsuggests that the putty-like consistency of such compositions offersdistinct advantages in moldability and workability.

Osteotech, Inc., of Shrewsbury, N.J., markets a bone graft substituteunder the mark Grafton®. It is comprised of demineralized bone matrixand glycerol as a carrier material. The carrier material, glycerol, is aviscous, gel-like, weak alcohol that is hydrophilic and water-soluble.It is recognized by the Food and Drug Administration as a “GenerallyRegarded As Safe” substance.

DePuy, Inc., of Warsaw, Ind., markets a bone graft substitute under themark DynaGraft®. It is comprised of demineralized bone matrix andpoloxamer as a carrier material. Poloxamer is a reverse phase polymerwhich becomes more viscous with increasing temperature.

Nothing in the known prior art discloses or suggests a bone graftsubstitute composition including calcium sulfate, a mixing solution suchas sterile water, and a plasticizing substance such ascarboxymethylcellulose, and having an extended set time and sufficientrobustness to withstand fluid impact with minimal erosion.

SUMMARY OF THE INVENTION

A basic concept of the present invention is to provide bone graftsubstitute composition having an extended set time and sufficientrobustness to withstand fluid impact with minimal erosion for expandedclinical applications.

The bone graft substitute composition of the present inventioncomprises, in general, calcium sulfate; a mixing solution such assterile water; and a plasticizing substance such ascarboxymethylcellulose.

One object of the present invention is to provide a bone graftsubstitute composition that can be mixed into a paste and then loadedinto a syringe and ejected for an extended period of time (e.g., morethan ten minutes).

Another object of the present invention is to provide a bone graftsubstitute composition that can be mixed into a putty and then handledand formed into desired shapes for an extended period of time (e.g.,more than ten minutes).

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE is a plot of displacement (mm) vs. time (sec) for certainembodiments of bone graft substitute compositions.

DETAILED DESCRIPTION

The bone graft substitute composition of the present inventioncomprises, in general, a quantity of calcium sulfate, a quantity offluid (e.g., sterile water), and a quantity of a plasticizing substance(e.g., methylcellulose) which provides a resultant composition that isrobust and has an extended set time. The extended set time of theresultant composition provides a useful working time of at least 5minutes to allow sufficient time for a surgeon to properly apply thebone graft substitute composition, while the robustness of the resultantcomposition allows the implanted composition to withstand the typicalpressure of body fluids, irrigation fluids and/or suctioning withminimal material erosion, disintegration or dissolution.

The bone graft substitute composition of the present invention maycomprise a mixture of calcium sulfate; a mixing solution selected fromthe group consisting of sterile water, inorganic salts, and cationicsurface active agents including sodium chloride, phosphate bufferedsaline, potassium chloride, sodium sulfate, potassium sulfate, EDTA,ammonium sulfate, ammonium acetate, and sodium acetate, etc.; and aplasticizing substance selected from the group consisting of cellulosederivatives including sodium carboxymethylcellulose, methylcellulose,hydroxypropyl methylcellulose, hydroxypropylcellulose, ethylcellulose,hydroxethylcellulose and cellulose acetate butyrate, and highermolecular weight alcohols including glycerol and vinyl alcohols, etc.The bone graft substitute composition may include demineralized bonematrix. One formulation of the composition may be approximately 100parts calcium sulfate by weight, 11.1 parts carboxymethylcellulose byweight, 185.2 parts water by weight, and 69.4 parts demineralized bonematrix by weight. Another formulation of the composition may beapproximately 100 parts calcium sulfate by weight, 6.3 partscarboxymethylcellulose by weight, and 31 parts water by weight. Anotherformulation of the composition may be approximately 100 parts calciumsulfate by weight, 1.2 parts carboxymethylcellulose by weight, and 31parts water by weight. Another formulation of the composition maybeapproximately 80-120 parts calcium sulfate by weight, 1-40 partscarboxymethylcellulose by weight, and 21-250 parts water by weight. Thecomposition may include a bioactive agent selected from the groupconsisting of demineralized bone matrix, growth factors, hyaluronicacid, bone morphogenic proteins, bone autograft, and bone marrow, etc.The composition may include sodium bicarbonate. For example, thecomposition may include 0.1-2% sodium bicarbonate by weight for creatinga porous structure in the resultant composition. Possible embodiments ofthe bone graft substitute composition of the present invention mayinclude at least one additive selected from the group consisting ofantiviral agent, antimicrobial agent, antibiotic agent, amino acid,peptide, vitamin, inorganic element, protein synthesis co-factor,hormone, endocrine tissue, synthesizer, enzyme, polymer cell scaffoldingagent with parenchymal cells, angiogenic drug, demineralized bonepowder, collagen lattice, antigenic agent, cytoskeletal agent,mesenchymal stem cells, bone digester, antitumor agent, cellularattractant, fibronectin, growth hormone, cellular attachment agent,immunosuppressant, nucleic acid, surface active agent, calcium phosphatematerials, such as hydroxyapatite or tricalcium phosphate, penetrationenhancer, bone allograft, and chunks, shards, and/or pellets of calciumsulfate.

Preferred Embodiment 1

An injectable bone graft substitute composition having the followingpreferred formulation: 100 parts by weight of medical grade calciumsulfate hemihydrate (MGCSH), 11.1 parts by weight ofcarboxymethylcellulose (CMC), 69.4 parts by weight of demineralized bonematrix (DBM), and 162 parts by weight of sterile water.

The preferred method for mixing this putty bone graft substitutecomposition comprises the following steps: (1) dry blend the powdercomponents (i.e., the calcium sulfate hemihydrate,carboxymethylcellulose, and demineralized bone matrix); (2) add thesterile water; and (3) mix or stir all components for approximately 30seconds to one minute or until the desired putty-like consistency isachieved.

The resultant injectable bone graft substitute composition has thefollowing characteristic/criteria:

Handability—the resultant composition should: (a) be a single cohesivebolus; (b) be able to be handled and manipulated with minimal to nomaterial transfer (sticking) to latex gloved hand; (c) be able to behandled without material crumbling or falling apart; and (d) exhibitminimal cracking or “tearing” with extreme manipulation, e.g., hardsqueezing;

Ejectability—the resultant composition should: (a) be able to be easilymanipulated, e.g., rolled into a long cylinder or other suitable shape,so as to be manually placed into an appropriate injection apparatus,e.g., a syringe; and (b) be able to be ejected through a ⅛ inch (0.3175centimeter) diameter orifice with relatively little pressure required;and

Robustness—the resultant composition, after being placed or injectedinto or onto the desired location, should be able to withstand bodyfluids, reasonable irrigation fluids and/or suctioning with minimalmaterial erosion, disintegration or dissolution.

Preferred Embodiment 2

A putty bone graft substitute composition having the following preferredformulation: 100 parts by weight of medical grade calcium sulfatehemihydrate (MGCSH), 11.1 parts by weight of carboxymethylcellulose(CMC), and 47 parts by weight of sterile water.

The preferred method for mixing this putty bone graft substitutecomposition comprises the following steps: (1) dry blend the powdercomponents (i.e., the calcium sulfate hemihydrate, andcarboxymethylcellulose); (2) add the sterile water; and (3) mix or stirall components for approximately 30 seconds to one minute or until thedesired putty-like consistency is achieved.

The resultant putty bone graft substitute composition has the followingcharacteristic/criteria:

Handability—the resultant composition should: (a) be a single cohesivebolus; (b) be able to be handled and manipulated with minimal to nomaterial transfer (sticking) to latex gloved hand; (c) be able to behandled without material crumbling or falling apart; and (d) exhibitminimal cracking or “tearing” with extreme manipulation, e.g., hardsqueezing; and

Robustness—the resultant composition, after being placed or injectedinto or onto the desired location, should be able to withstand bodyfluids, reasonable irrigation fluids and/or suctioning with minimalmaterial erosion, disintegration or dissolution.

Preferred Embodiment 3

A paste bone graft substitute composition having the following preferredformulation: 100 parts by weight of medical grade calcium sulfatehemihydrate (MGCSH), 1.2 parts by weight of carboxymethylcellulose(CMC), and 31 parts by weight of sterile water.

The preferred method for mixing this putty bone graft substitutecomposition comprises the following steps: (1) dry blend the powdercomponents (i.e., the calcium sulfate hemihydrate, andcarboxymethylcellulose); (2) add the sterile water; and (3) mix or stirall components for approximately 30 seconds to one minute or until thedesired putty-like consistency is achieved.

The resultant paste bone graft substitute composition has the followingcharacteristic/criteria:

Ejectability—the resultant composition should be able to be ejectedthrough a ⅛ inch (0.3175 centimeter)diameter orifice with relativelylittle pressure required.

Preferred Embodiment 4

A bone graft substitute composition having the following preferredformulation: approximately 80-120 parts medical grade calcium sulfatehemihydrate by weight; approximately 21-250 parts sterile water byweight; and approximately 1-40 parts sodium carboxymethylcellulose byweight. This preferred formulation may include approximately 10-100parts demineralized bone matrix by weight.

The preferred method for mixing this bone graft substitute compositioncomprises the following steps: (1) dry blend the powder components(i.e., the calcium sulfate hemihydrate, and sodiumcarboxymethylcellulose, and, if included, the demineralized bonematrix); (2) add the sterile water; and (3) mix or stir all componentsfor approximately 30 seconds to one minute or until the desiredconsistency is achieved.

The resultant bone graft substitute composition has the followingcharacteristic/criteria:

Handability—the resultant composition should: (a) be a single cohesivebolus; (b) be able to be handled and manipulated with minimal to nomaterial transfer (sticking) to latex gloved hand; (c) be able to behandled without material crumbling or falling apart; and (d) exhibitminimal cracking or “tearing” with extreme manipulation, e.g., hardsqueezing;

Ejectability—the resultant composition should: (a) be able to be easilymanipulated, e.g., rolled into a long cylinder or other suitable shape,so as to be manually placed into an appropriate injection apparatus,e.g., a syringe; and (b) be able to be ejected through a ⅛ inch (0.3175centimeter) diameter orifice with relatively little pressure required;and

Robustness—the resultant composition, after being placed or injectedinto or onto the desired location, should be able to withstand bodyfluids, reasonable irrigation fluids and/or suctioning with minimalmaterial erosion, disintegration or dissolution.

Preferred Embodiment 5

In some preferred embodiments, the bone graft substitute compositionincludes calcium sulfate, e.g., calcium sulfate hemihydrate; a mixingsolution, e.g., sterile water; and a plasticizing substance, e.g.,hyaluronic acid or a cellulose derivative such as methylcellulose. Theplasticizing substance can include, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, ethylcellulose, hydroxyethylcellulose,and/or cellulose acetate butyrate. The mixing solution can include, forexample, sterile water, inorganic salt, cationic surface active agent,bone marrow aspirate, platelet concentrate, blood, pharmaceuticaladditives in solution, or combinations of these materials. Examples ofadditives are medicaments or pesticides. Examples of medicaments areantibiotics, chemotherapeutic agents, growth factors, and analgesics.Examples of antibiotics are tetracycline hydrochloride, vancomycin,cephalosporins, and aminoglycocides such as tobramycin and gentamicin.Examples of chemotherapeutic agents are cis-platinum, ifosfamide,methotrexate, and doxorubicin hydrochloride (Adriamycin®). Examples ofgrowth factors are transforming growth factor beta (TGF-Beta), bonemorphogenic protein (BMP), basic fiberblast growth factor,platelet-derived growth factor, and other polypeptide growth factors.Examples of analgesics are anesthetics such as lidocaine hydrochloride(Xylocaine®), bipivacaine hydrochloride (Marcaine®), and non-steroidalanti-inflammatory drugs such as ketorolac tromethamine (Toradol®). Thecationic surface active agent can include sodium chloride, phosphatebuffered saline, potassium chloride, sodium sulfate, ammonium sulfate,ammonium acetate, or sodium acetate.

Generally, the bone graft substitute composition includes a given amountof calcium sulfate, e.g., normalized to 100 parts by weight ofCaSO₄.½H₂O; an amount of the plasticizing substance sufficient toprovide a good biological response, e.g., about 1 to about 10 parts, orabout 1 to about 7 parts, or about 2 to about 6 parts, by weight; and asufficient amount of the mixing solution to provide good handability,e.g., about 20 to about 40 parts, or about 20 to about 35 parts, byweight, such that the composition can be conveniently handled andshaped. For a bone graft composition having CaSO₄. ½H₂O and sterilewater as the mixing solution, specific compositions are a function ofthe plasticizing substance used in the composition.

In an embodiment having hydroxypropyl cellulose (HPC) as theplasticizing substance, the composition can include 100 parts by weightof calcium sulfate, e.g., CaSO₄. ½H₂O; about 2 to about 7 parts, e.g.,about 3 parts, by weight of HPC; and about 26 to about 32 parts, e.g.,about 28 parts, by weight of the mixing solution, e.g., sterile water.

In an embodiment having hydroxypropyl methyl cellulose (HPMC) as theplasticizing substance, the composition can include 100 parts by weightof calcium sulfate, e.g., CaSO₄. ½H₂O; about 1 to about 6 parts, e.g.,about 2 parts, by weight of HPMC; and about 23 to about 32 parts, e.g.,about 25 parts, by weight of the mixing solution, e.g., sterile water.

In an embodiment having hyaluronic acid as the plasticizing substance,the composition can include 100 parts by weight of calcium sulfate,e.g., CaSO₄. ½H₂O; about 4 to about 6 parts, e.g., about 5 parts, byweight of hyaluronic acid; and about 23 to about 40 parts, e.g., about30 to about 35 parts, by weight of the mixing solution, e.g., sterilewater.

In yet another embodiment, the composition can include about 100 partsby weight of the calcium sulfate hemihydrate, about 25 to about 65 partsby weight of the mixing solution, e.g., water, and about 1.5 to about 8parts by weight of methylcellulose. For example, the composition caninclude about 100 parts by weight of the calcium sulfate, e.g., calciumsulfate hemihydrate, about 33.6 parts by weight of the mixing solution,e.g., water, and about 5.25 parts by weight of methylcellulose.

The compositions are formed according to the methods described above.Powder components (e.g., calcium sulfate and plasticizing substance) aredry blended. The mixing solution, e.g., water, is added to the powdercomponents, e.g., prior to use in the operating room, and the mixture ismixed or stirred for about 30-60 seconds or until a desired consistencyis achieved.

The resulting bone graft substitute composition is a paste or puttyhaving similar handability, ejectability and/or robustness as describedabove.

The substitute composition can be handled and shaped such that it can beconveniently positioned and secured into a surgical site. Thecomposition can set up relatively hard, e.g., it can be used as aninteroperative structural support, it can be resistant to substantialcollapse, or it can withstand fluid impact without substantial erosion.The substitute composition has relatively low to no risk of transmittinginfectious disease because, for example, it does not include biologicalmaterials such as materials from a cadaver. The composition isrelatively inexpensive to produce.

The resulting bone graft substitute can also be used as a carrier, forexample, by mixing it with other materials, such as, for example,allografts, antibiotics, growth factors, cancellous bone chips, orsynthetically derived or naturally derived chips of minerals such ascalcium phosphate or calcium carbonate. This can provide the compositionwith versatility and flexibility by allowing a user to formulate a mixedcomposition according to a desired application.

Preferred Embodiment 6

Other preferred embodiments of a bone graft substitute compositioninclude calcium sulfate, e.g., calcium sulfate hemihydrate,demineralized bone matrix, allograft materials, preferably, cancellousbone chips (an osteoconductive substrate) from a cadaver sufficient toprovide a desired texture, a plasticizing substance sufficient toprovide a good biological response, and a mixing solution sufficient toprovide a good handability. The plasticizing substance can includecarboxymethylcellulose, e.g., sodium carboxymethylcellulose,methylcellulose, hydroxypropyl methyl cellulose, ethylcellulose,hydroxyethylcellulose, and/or cellulose acetate butyrate. The mixingsolution can include, for example, sterile water, inorganic salt,cationic surface active agent, bone marrow aspirates, plateletconcentrates, blood, pharmaceutical additives, or combinations of thesematerials. Examples of additives are listed in Preferred Embodiment 5.The cationic surface active agent can include sodium chloride, phosphatebuffered saline, potassium chloride, sodium sulfate, ammonium sulfate,ammonium acetate, or sodium acetate. The cancellous bone can have aparticle size of about 1-10 mm, e.g., about 1-6.7 mm, about 1-5.6 mm,about 1-4 mm, or about 4-10 mm.

Generally, the composition can include about 80 to about 120 parts byweight of calcium sulfate, about 10 to about 100 parts by weight ofdemineralized bone matrix, about 10 to about 100 parts by weight ofallograft materials, about 1 to about 40 parts by weight of aplasticizing substance, and about 21 to about 250 parts by weight of amixing solution. Preferably, the composition includes about 90 to about110 parts by weight of calcium sulfate, about 10 to about 50 parts byweight of demineralized bone matrix, about 15 to about 50 parts byweight of cancellous bone, about 5 to about 20 parts by weight of aplasticizing substance, and about 80 to about 120 parts by weight of amixing solution. More preferably, the composition includes about 98 toabout 102 parts by weight of calcium sulfate, about 13 to about 23 partsby weight of demineralized bone matrix, about 24 to about 33 parts byweight of cancellous bone, about 15 to about 20 parts by weight of aplasticizing substance, and about 95 to about 105 parts by weight of amixing solution. Most preferably, the composition includes about 100parts by weight of calcium sulfate, about 18 to about 19 parts, e.g.,about 18 parts, by weight of demineralized bone matrix, about 27 toabout 28 parts, e.g., about 27.6 parts, by weight of cancellous bone,about 17 to about 18 parts, e.g., about 17.6 parts, by weight of aplasticizing substance, and about 101 to about 102 parts, e.g., about101 parts, by weight of a mixing solution.

The compositions are formed according to the methods described above.Powder components (e.g., calcium sulfate hemihydrate, demineralized bonematrix, cancellous bone chips, and carboxymethylcellulose) are dryblended. The mixing solution, e.g., water, is added to the powdercomponents, and the mixture is mixed or stirred for about 30-60 secondsor until a desired consistency is achieved.

In some embodiments, the calcium sulfate, demineralized bone matrix, andthe plasticizing substance can be packaged together in a firstcontainer, e.g., a vial; and the allograft material, e.g., cancellousbone chip, can be packaged in a second container. A user, e.g., asurgeon, can customize a composition by blending the allograft materialwith the contents of the first vial and the mixing solution to form acomposition with the desired texture.

In some circumstances, the first container can contain a predeterminedamount of allograft material, e.g., about 10 parts of cancellous bonechips; and the second container can contain a predetermined amount ofallograft material, e.g., up to about 90 parts of cancellous bone chips.In addition to allowing the user to form a composition by blending themixing solution with only the contents of first container, this allowsthe user to blend additional allograft material into the composition toa desired texture.

The resulting bone graft substitute composition is a paste or puttyhaving similar handability, ejectability and/or robustness as describedabove. The cancellous bone chips can provide the composition with goodstructural support, and the relatively large surface area of thecancellous bone chips can provide the composition with goodosteoconduction.

Preferred Embodiment 7

Other preferred embodiments of a bone graft substitute compositioninclude calcium sulfate, e.g., calcium sulfate hemihydrate,demineralized bone matrix, allograft materials, preferably, cancellousbone chips (an osteoconductive substrate) from a cadaver sufficient toprovide a desired texture, a plasticizing substance sufficient toprovide a good biological response, and a mixing solution sufficient toprovide a good handability. The plasticizing substance can includecarboxymethylcellulose, e.g., sodium carboxymethylcellulose,methylcellulose, hydroxypropyl methyl cellulose, ethylcellulose,hydroxyethylcellulose, and/or cellulose acetate butyrate. The mixingsolution can include, for example, sterile water, inorganic salt,cationic surface active agent, bone marrow aspirates, plateletconcentrates, blood, pharmaceutical additives, or combinations of thesematerials. Examples of additives are listed in Preferred Embodiment 5.The cationic surface active agent can include sodium chloride, phosphatebuffered saline, potassium chloride, sodium sulfate, ammonium sulfate,ammonium acetate, or sodium acetate. The cancellous bone can have aparticle size of about 1-10 mm, e.g., about 1-4 mm.

Generally, the composition can include about 80 to about 120 parts byweight of calcium sulfate, about 10 to about 100 parts by weight ofdemineralized bone matrix, about 20 to about 130 parts by weight ofallograft materials, e.g., cancellous bone, about 1 to about 40 parts byweight of a plasticizing substance, and about 21 to about 250 parts byweight of a mixing solution. Preferably, the composition includes about90 to about 110 parts by weight of calcium sulfate, about 10 to about 50parts by weight of demineralized bone matrix, about 15 to about 50 partsby weight of allograft materials, about 5 to about 20 parts by weight ofa plasticizing substance, and about 80 to about 120 parts by weight of amixing solution. More preferably, the composition includes about 98 toabout 102 parts by weight of calcium sulfate, about 13 to about 23 partsby weight of demineralized bone matrix, about 37 to about 46 parts byweight of allograft materials, about 15 to about 20 parts by weight of aplasticizing substance, and about 95 to about 105 parts by weight of amixing solution. Most preferably, the composition includes about 100parts by weight of calcium sulfate, about 18 to about 19 parts, e.g.,about 18 parts, by weight of demineralized bone matrix, about 40 toabout 42 parts, e.g., about 41 parts, by weight of allograft materials,about 17 to about 18 parts, e.g., about 17.6 parts, by weight of aplasticizing substance, and about 101 to about 102 parts, e.g., about101 parts, by weight of a mixing solution.

The compositions are formed according to the methods described above.Powder components (e.g., calcium sulfate hemihydrate, demineralized bonematrix, cancellous bone chips, and carboxymethylcellulose) are dryblended. The mixing solution, e.g., water, is added to the powdercomponents, and the mixture is mixed or stirred for about 30-60 secondsor until a desired consistency is achieved.

In some embodiments, the calcium sulfate, demineralized bone matrix, andthe plasticizing substance can be packaged together in a firstcontainer, e.g., a vial; and the allograft material, e.g., cancellousbone chip, can be packaged in a second container. A user, e.g., asurgeon, can customize a composition by blending the allograft materialwith the contents of the first vial and the mixing solution to form acomposition with the desired texture.

In some circumstances, the first container can contain a predeterminedamount of allograft material, e.g., about 10 parts of cancellous bonechips; and the second container can contain a predetermined amount ofallograft material, e.g., up to about 90 parts of cancellous bone chips.In addition to allowing the user to form a composition by blending themixing solution with only the contents of first container, this allowsthe user to blend additional allograft material into the composition toa desired texture.

The resulting bone graft substitute composition is a paste or puttyhaving similar handability, ejectability and/or robustness as describedabove. The cancellous bone chips can provide the composition with goodstructural support, and the relatively large surface area of thecancellous bone chips can provide the composition with goodosteoconduction.

Preferred Embodiment 8

Still other preferred embodiments of a bone graft substitute compositioninclude calcium sulfate, e.g., calcium sulfate hemihydrate,demineralized bone matrix, allograft materials, preferably, cancellousbone chips (an osteoconductive substrate) from a cadaver sufficient toprovide a desired texture, a plasticizing substance sufficient toprovide a good biological response, and a mixing solution sufficient toprovide a good handability. The plasticizing substance can includecarboxymethylcellulose, e.g., sodium carboxymethylcellulose,methylcellulose, hydroxypropyl methyl cellulose, ethylcellulose,hydroxyethylcellulose, and/or cellulose acetate butyrate. The mixingsolution can include, for example, sterile water, inorganic salt,cationic surface active agent, bone marrow aspirates, plateletconcentrates, blood, pharmaceutical additives, or combinations of thesematerials. Examples of additives are listed in Preferred Embodiment 5.The cationic surface active agent can include sodium chloride, phosphatebuffered saline, potassium chloride, sodium sulfate, ammonium sulfate,ammonium acetate, or sodium acetate. The cancellous bone can have aparticle size of about 1-10 mm, e.g., about 4-10 mm, or about 4-9.5 mm.Without wishing to be bound to theory, it is believed that usingcancellous bone of increased particle size provides the composition withenhanced capacity for fluid absorption. As a result, the composition canbe loaded with a relatively large amount of the mixing solution, such asplatelet concentrates or bone marrow aspirates, that can be delivered toa surgical site. In some embodiments, the powder components of thecomposition can absorb up to a 1:1 ratio of mixing solution by volume.

Generally, the composition can include about 80 to about 120 parts byweight of calcium sulfate, about 10 to about 120 parts by weight ofdemineralized bone matrix, about 20 to about 150 parts by weight ofallograft materials, e.g., cancellous bone, about 1 to about 40 parts byweight of a plasticizing substance, and about 50 to about 300 parts byweight of a mixing solution. Preferably, the composition includes about90 to about 110 parts by weight of calcium sulfate, 10 to about 60 partsby weight of demineralized bone matrix, about 40 to about 120 parts byweight of allograft materials, about 5 to about 20 parts by weight of aplasticizing substance, and about 140 to about 180 parts by weight of amixing solution. More preferably, the composition includes about 98 toabout 102 parts by weight of calcium sulfate, about 25 to about 35 partsby weight of demineralized bone matrix, about 44 to about 110 parts byweight of allograft materials, about 15 to about 20 parts by weight of aplasticizing substance, and about 160 to about 170 parts by weight of amixing solution. Most preferably, the composition includes about 100parts by weight of calcium sulfate, about 30 to about 32 parts, e.g.,about 31 parts, by weight of demineralized bone matrix, about 99 toabout 105 parts, e.g., about 102 parts, by weight of allograftmaterials, about 17 to about 18 parts, e.g., about 17.6 parts, by weightof a plasticizing substance, and about 162 to about 168 parts, e.g.,about 164 parts, by weight of a mixing solution.

The compositions are formed according to the methods described above.Powder components (e.g., calcium sulfate hemihydrate, demineralized bonematrix, cancellous bone chips, and carboxymethylcellulose) are dryblended. The mixing solution, e.g., water, is added to the powdercomponents, and the mixture is mixed or stirred for about 130-60 secondsor until a desired consistency is achieved.

In some embodiments, the calcium sulfate, demineralized bone matrix, andthe plasticizing substance can be packaged together in a firstcontainer, e.g., a vial; and the allograft material, e.g., cancellousbone chip, can be packaged in a second container. A user, e.g., asurgeon, can customize a composition by blending the allograft materialwith the contents of the first vial and the mixing solution to form acomposition with the desired texture.

In some circumstances, the first container can contain a predeterminedamount of allograft material, e.g., about 45 parts of cancellous bonechips; and the second container can contain a predetermined amount ofallograft material, e.g., up to about 150 parts of cancellous bonechips. In addition to allowing the user to form a composition byblending the mixing solution with only the contents of first container,this allows the user to blend additional allograft material into thecomposition to a desired texture. The amount of allograft material inthe first and second containers can vary within the ranges providedabove.

The resulting bone graft substitute composition is a paste or puttyhaving similar handability, ejectability and/or robustness as describedabove. The cancellous bone chips can provide the composition with goodstructural support, and the relatively large surface area of thecancellous bone chips can provide the composition with goodosteoconduction.

Preferred embodiments 5-8 can be packaged as kits that allow the user tocustomize a desired composition by combining the mixing solution withthe desired powder components according to a desired use, texture,consistency, and/or concentration of a particular component, such ascancellous bone.

Tests

The majority of tests done to date on the bone graft substitutecomposition of the present invention basically consist of mixing aspecific formulation and then assessing and recording the mixing,handling, consistency, and injectability properties of the resultantmaterial.

Formulation Tests

Injectable Bone Graft Substitute Composition: Formulations with varioustypes and amounts of carboxymethylcellulose and demineralized bonematrix have been tested. Specific examples include: (1)carboxymethylcellulose percentages of 1-10% by weight; (2) types ofcarboxymethylcellulose have included high viscosity, medium viscosity,and low viscosity from 3 vendors (e.g., Aqualon® 7HF PH sodiumcarboxymethylcellulose from Hercules Incorporated, Hercules Plaza, 1313North Market Street, Wilmington, Del. 19894-0001); (3)carboxymethylcellulose sterilized by gamma or electronic beamsterilization (medium and low doses); (4) demineralized bone matrixpercentages up to 65% by volume; (5) differently processed demineralizedbone matrix, air dried and freeze dried; (6) demineralized bone matrixfrom two vendors (e.g., human freeze dried demineralized bone matrixfrom AlloSource, 8085 E. Harvard Ave., Denver, Colo. 80231); and (7)animal demineralized bone matrix, including bovine and canine.

For all these formulations, varying amounts of water, between 31-200parts by weight, have been tested. The mixing, handling, consistency,and injectability properties were assessed and formulas chosen such thatthey met the mixing, handability, ejectability, and robustnesscharacteristics/criteria stated hereinabove.

Paste And Putty Bone Graft Substitute Composition: These were the firsttests done and included formulations with compositions having 100 partsby weight medical grade calcium sulfate hemihydrate, and between 1-10%by weight carboxymethylcellulose, and between 31-200 parts by weightwater. As was the case with the injectable bone graft substitutecomposition, mixing, handability, consistency, injectability, androbustness properties were assessed for the different formulations.Specific tests have included: (1) varying the carboxymethylcellulosepercentages from 0.25% up to 10% by weight, (2) using inorganic saltsolutions including 2% sodium chloride (NaCl) by weight, 2-4% sodiumsulfate (Na₂SO₄) by weight, and 2% potassium chloride (KCl) by weight.

As with the injectable bone graft substitute composition, varyingamounts of water, 31-200 parts by weight, were used.

EXAMPLE 1

The osteoinductive properties of the injectable bone graft substitutecomposition have been studied using an athymic mouse-intramuscularimplantation model. This animal model is widely accepted as the “goldstandard” for assessing osteoinductive characteristics of bone graftmaterials. In this model, a given amount of material is surgicallyplaced into a muscular site. After an implantation period of four weeks,the osteoinductive response is assessed using various analyticalmethods, including radiography, biochemical analysis (alkalinephosphatase levels and calcium content), and histomorphometry.

In this study, four athymic (nude) male mice (Harlan Sprague Dawley,Inc.) were used for each material group. Two muscle pouches were formedin the right and left gluteal muscles of each mouse and implanted witheither: (1) pellets which were manufactured using the composition givenin Preferred Embodiment 1, or (2) twenty (20) 5 mg of demineralized bonematrix which had been rehydrated with isotonic saline (0.9% NaCl). Thepellets made from Preferred Embodiment 1 were 3.0 mm in diameter, 2.5 mmin height and 25 mg in weight.

After twenty-eight (28) days the animals were sacrificed and thematerials explanted. The explants were analyzed for osteoinductivepotential by assessing the alkaline phosphatase activity and for newbone growth by histomorphometric analysis of histologic sections.

Samples to be analyzed for alkaline phosphatase activity were minced,sonicated, and extracted with water saturated butanol. The extracts wereassayed for protein content using a Pierce BCA Protein Assay Kit (PierceChemical Co.) and measuring the conversion of para-nitrophenylphosphate(PNPP) to para-nitrophenol (pNP) with time. The results were expressedas μmole pNP formed/min/μg tissue protein.

Samples intended for histomorphometric analyses were prepared usingstandard histological procedures. The percent viable bone (new boneformation) was quantitated employing computer software (Adobe Photo Shop3.0.4 and HNIH 1.61), in conjunction with a microscope equipped with avideo camera. Data was reported as percent viable bone relative to thetotal cross-sectional area analyzed.

The alkaline phosphatase levels (μmole pNP formed/min/μg tissue protein)and percent viable bone results for the groups of mice implanted withDBM only and with injectable putty manufactured using the compositiongiven in Preferred Embodiment 1 are shown in Table 1.

TABLE 1 Osteoinductive Results Alkaline Phosphatase Levels and PercentViable Bone Alkaline Phosphatase Levels (μmole pNP formed/ PercentViable Bone Group min/μg tissue protein) (%) DMB only 2.1 × 10⁻⁵ ± 0.3 ×10⁻⁵ 6.5% ± 1.0% Injectable Putty 3.0 × 10⁻⁵ ± 0.2 × 10⁻⁵ 4.7% ± 0.9%(Preferred Embodiment 1)

EXAMPLE 2

A study was performed on canines to evaluate healing of bone defectsusing materials with the composition given in Preferred Embodiment 1.The DBM used in these compositions was fresh frozen canine DBM(Veterinarian Transplant Services, Seattle, Wash.). Two methods wereused to produce the test materials. The first material group consistedof a blend of DBM, calcium sulfate, and CMC powder that was irradiatedsterilized, while the second group mixed canine DBM with the calciumsulfate-CMC blend at the time of surgery.

In this canine animal model, large medullary cylindrical defects (13 mmdiameter ×50 mm length)were created bilaterally in the proximal humeriby drilling axially through the greater tubercle. Six to 7 cc of testmaterial were injected into prepared cavities using a large-borecatheter-tip syringe. Left humeri received the premixed material thathand been sterilized and the right humeri received the material mixedintraoperatively which utilized non-irradiated canine DBM. Radiographsof the humeri were obtained preoperative, immediately postoperative, andat 2, 3, and 6 weeks. Following euthanasia after 6 weeks, the explantedhumeri were sectioned transversely, radiographed, and processed forplastic imbedded undecalcified histology. The histologic sections werestained with basic fuchsin and toluidine blue and examined by lightmicroscopy.

Post-operative radiographs revealed all test materials to be wellcontained in the prepared cavities. Normal would healing occurred andthere were no postoperative infections. Serial clinical radiographsshowed a progressive decrease in materials density with time. Nodifference was evident between the right and left sides.

Contact radiographs of the cut sections demonstrated no difference inpattern or density of bone filling the right and left defects,non-irradiated and irradiated canine DBM materials groups, respectively.Serial sections for all the dogs showed between 30-100% filling of thedefect, with one dog showing almost complete filling for all sections.

Histologically, the nature of new bone formation and the amount ofresidual material were similar in the right and left defects. In theperipheral one-third of the defects, new bone was present at the marginsand haversian surfaces of abundant DBM particles. Residual calciumsulfate was evident, incorporated within slender bone trabeculae,independent of DBM particles. New bone formation in the central aspectof the defects was more variable, with some vascular fibrosus tissueshown. No foreign body or inflammatory response was seen in any of theslides, indicating that the materials had extremely goodbiocompatibilty.

Thus, materials with compositions given in Preferred Embodiment 1 wereshown to be well tolerated by the bone and to heal a large medullarydefect 30-100% at six 20 weeks with viable new bone in a canine bonedefect model.

EXAMPLE A

The following samples were prepared as described above to determinetheir ability to resist compression through a mesh.

Sample A Sample B Sample C CaSO₄•½H₂O 100 100 100 Demineralized BoneMatrix 69.4 18 31 Cancellous Bone Chips — 41 102 (1-4 mm) (4-10 mm)Carboxymethylcellulose 11.1 17.6 17.6 Sterile Water 162 101 164

Approximately 10 cc of each sample was placed into a cylinder (about 22mm in diameter) formed of a mesh (about 2.7 mm grid). The sample waspacked in the cylinder to a height of about 29.5-34 mm.

The sample was loaded into a mechanical testing system (Model 1331Testing System, available from Instron (Canton, Mass.)) with thecylinder oriented vertically. A ram (a 20 mm diameter flat end rod) wasplaced in the cylinder on top of the sample. A 100 N load was applied inone second. Displacement data were recorded every 0.1 sec (usingNotebookPro software, available from Labtech (Andover, Mass.)). FIG. 1shows the displacement (mm) of Samples A, B, and C (n=1) as a functionof time (sec). The samples displayed good resistance to compression. Asa result, in some circumstances, the samples may be more likely to beretained in a surgical site, e.g., a cavity, after they are positionedin place.

Although the present invention has been described and illustrated withrespect to preferred embodiments and preferred uses therefor, it is notto be so limited since modifications and changes can be made thereinwhich are within the full intended scope of the invention. For example,in embodiments that include cancellous bone, calcium phosphatematerials, such as hydroxyapatite or tricalcium phosphate, can besubstituted for all or a portion of the cancellous bone.

Other embodiments are within the claims.

1. A bone graft substitute composition, comprising: about 80 to about120 parts by weight of calcium sulfate; about 10 to about 100 parts byweight of demineralized bone matrix; about 20 to about 130 parts byweight of cancellous bone; about 1 to about 40 parts by weight of aplasticizing substance; and about 21 to about 250 parts by weight of amixing solution, wherein the cancellous bone has a particle size betweenabout 1 and about 4 mm.
 2. The composition of claim 1, wherein thecalcium sulfate comprises calcium sulfate hemihydrate.
 3. Thecomposition of claim 1, wherein the plasticizing substance comprises acellulose derivative.
 4. The composition of claim 1, wherein theplasticizing substance is selected from a group consisting of sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methyl cellulose,hydroxypropyl cellulose, ethylcellulose, hydroxyethylcellulose, andcellulose acetate butyrate.
 5. The composition of claim 1, wherein themixing solution is selected from a group consisting of sterile water,inorganic salt, and cationic surface active agent.
 6. The composition ofclaim 5, wherein the cationic surface active agent is selected from agroup consisting of sodium chloride, phosphate buffered saline,potassium chloride, sodium sulfate, ammonium sulfate, ammonium acetate,and sodium acetate.
 7. The composition of claim 1, wherein the mixingsolution comprises sterile water.
 8. The composition of claim 1, whereinthe mixing solution comprises a material selected from a groupconsisting of bone marrow aspirate, platelet concentrate, blood, anantibiotic, a chemotherapeutic agent, a growth factor, and an analgesic.9. The composition of claim 1, comprising: about 90 to about 110 partsby weight of calcium sulfate; about 10 to about 50 parts by weight ofdemineralized bone matrix; about 15 to about 50 parts by weight ofcancellous bone; about 5 to about 20 parts by weight of a plasticizingsubstance; and about 80 to about 120 parts by weight of a mixingsolution.
 10. The composition of claim 1, comprising: about 98 to about102 parts by weight of calcium sulfate; about 13 to about 23 parts byweight of demineralized bone matrix; about 37 to about 46 parts byweight of cancellous bone; about 15 to about 20 parts by weight of aplasticizing substance; and about 95 to about 105 parts by weight of amixing solution.
 11. The composition of claim 1, comprising: about 100parts by weight of calcium sulfate; about 18 to about 19 parts by weightof demineralized bone matrix; about 40 to about 42 parts by weight ofcancellous bone; about 17 to about 18 parts by weight of a plasticizingsubstance; and about 101 to about 102 parts by weight of a mixingsolution.
 12. A bone graft substitute composition, comprising: about 80to about 120 parts by weight of calcium sulfate; about 10 to about 120parts by weight of demineralized bone matrix; about 20 to about 150parts by weight of cancellous bone; about 1 to about 40 parts by weightof a plasticizing substance; and about 50 to about 300 parts by weightof a mixing solution, wherein the cancellous bone has a particle sizebetween about 4 and about 10 mm.
 13. The composition of claim 12,wherein the calcium sulfate comprises calcium sulfate hemihydrate. 14.The composition of claim 12, wherein the plasticizing substancecomprises a cellulose derivative.
 15. The composition of claim 12,wherein the plasticizing substance is selected from a group consistingof sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethylcellulose,hydroxyethylcellulose, and cellulose acetate butyrate.
 16. Thecomposition of claim 12, wherein the mixing solution is selected from agroup consisting of sterile water, inorganic salt, and cationic surfaceactive agent.
 17. The composition of claim 16, wherein the cationicsurface active agent is selected from a group consisting of sodiumchloride, phosphate buffered saline, potassium chloride, sodium sulfate,ammonium sulfate, ammonium acetate, and sodium acetate.
 18. Thecomposition of claim 12, wherein the mixing solution comprises sterilewater.
 19. The composition of claim 12, wherein the mixing solutioncomprises a material selected from a group consisting of bone marrowaspirate, platelet concentrate, blood, an antibiotic, a chemotherapeuticagent, a growth factor, and an analgesic.
 20. The composition of claim12, comprising: about 90 to about 110 parts by weight of calciumsulfate; about 10 to about 60 parts by weight of demineralized bonematrix; about 40 to about 120 parts by weight of cancellous bone; about5 to about 20 parts by weight of a plasticizing substance; and about 140to about 180 parts by weight of a mixing solution.
 21. The compositionof claim 12, comprising: about 98 to about 102 parts by weight ofcalcium sulfate; about 25 to about 35 parts by weight of demineralizedbone matrix; about 44 to about 110 parts by weight of cancellous bone;about 15 to about 20 parts by weight of a plasticizing substance; andabout 160 to about 170 parts by weight of a mixing solution.
 22. Thecomposition of claim 12, comprising: about 100 parts by weight ofcalcium sulfate; about 30 to about 32 parts by weight of demineralizedbone matrix; about 99 to about 105 parts by weight of cancellous bone;about 17 to about 18 parts by weight of a plasticizing substance; andabout 162 to about 168 parts by weight of a mixing solution.
 23. A kitfor making a bone graft substitute composition, the kit comprising: afirst portion comprising: about 80 to about 120 parts by weight ofcalcium sulfate; about 10 to about 120 parts by weight of demineralizedbone matrix; a first portion of cancellous bone having a particle sizebetween about 4 and about 10 mm; and about 1 to about 40 parts by weightof a plasticizing substance; a second portion comprising: a secondportion of cancellous bone having a particle size between about 4 andabout 10 mm; and a third portion comprising: about 50 to about 300 partsby weight of a mixing solution, wherein the first and second portions ofcancellous bone total about 20 to about 150 parts by weight ofcancellous bone.
 24. The kit of claim 23, wherein the first portioncomprises about 40 to about 50 parts by weight of cancellous bone.
 25. Amethod of preparing a bone graft substitute composition, the methodcomprising: providing a first portion comprising: about 80 to about 120parts by weight of calcium sulfate; about 10 to about 120 parts byweight of demineralized bone matrix; a first portion of cancellous bonehaving a particle size between about 4 and about 10 mm; and about 1 toabout 40 parts by weight of a plasticizing substance; providing a secondportion comprising: a second portion of cancellous bone having aparticle size between about 4 and about 10 mm, wherein the first andsecond portions of cancellous bone total about 20 to about 150 parts byweight of cancellous bone; providing a third portion comprising: about50 to about 300 parts by weight of a mixing solution; and mixing thethird portion with the first portion to form a bone graft substitutecomposition.
 26. The method of claim 25, further comprising mixing aportion of the second portion with the third and first portions.